Systems and methods of creating a biofilm for the reduction of water contamination

ABSTRACT

In some embodiments, a system may reduce contaminants in water. A system may include a biofilm in a container. The biofilm may be formed from one or more bacteria coupled to one or more substrates. The bacteria may be selected to maximize the reduction of contaminants in water. The system may include one or more bacteria generators to provide bacteria to the biofilm and/or one or more air sources to provide an air bubble stream to the container and/or the bacteria generator. In some embodiments, bacteria may be preserved in a starvation phase. Bacteria may be incubated until they reach a starvation phase. The bacteria may then be preserved as beads or immobilized on a substrate. The preserved bacteria may be used in a system for the reduction of contaminants in water.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Contract #F41624-02-Z-9000 awarded by the Air Force AFIOH TRIES CollaborativeWater/Wastewater Treatment Technology Project. The Government hascertain rights to this invention.

BACKGROUND

1. Field of the Invention

The present invention relates to systems and methods for treating waterand preserving bacteria. More particularly, the invention relates to thereduction of contaminants from wastewater and preserving bacteria in astarvation phase.

2. Description of Related Art

Fluidized bed bioreactors are often used to treat wastewater. Granularmedia containing bacteria may be positioned in a water column andfluidization may be obtained by liquid recirculation or by external gasfed into the reactor. A biomass may form on the granular media.Wastewater may be batch processed by the biomass. Some new wastewatertreatment systems rely on activated sludge from operational wastewatertreatment systems to form a biofloc or biomass. However, formation ofthe biofloc or biomass from the sludge may be time consuming and may notallow customization for specific wastewater contaminants. In addition,as bacteria in a biofloc or biomass fall off of the mass and/or die, asystem to replenish specific strains of bacteria in the biofloc may notexist.

Bacteria are usually preserved in the logarithmic growth stage since thegrowth rate of bacteria exceeds the death rate during this stage. Thebacteria are usually preserved by lyophillization or by formingcompression tablets. These techniques are time consuming, inefficient,prone to contamination, and/or not cost effective.

SUMMARY

In some embodiments, a system for the reduction of contaminants in watermay include one or more containers that include bacteria coupled to oneor more substrates. In an embodiment, a biofilm may form in thecontainer that includes bacteria coupled to one or more substrates. Thesystem may include one or more bacteria generators that provide bacteriato a container. The system may also include one or more air sources toprovide air, as for example, an air bubble stream. In an embodiment, anair source may be coupled to containers and/or bacteria generators.

In some embodiments, the system may be used to process and/or reduce theamount of contaminants in wastewater. In certain embodiments, when asystem for the reduction of contaminants has foaming, one or morehydrophobic substrates may be added to at least one of the containerswith foaming. The bacteria may couple to the substrate. When thebacteria couple to the substrate, foaming may be reduced or eliminated.

In some embodiments, a biofilm may include one or more primary adhererbacteria and one or more secondary adherer bacteria. Primary adhererbacteria may couple to one or more substrates. In certain embodiments,primary adherer bacteria may also couple to one or more other bacteria.Secondary adherer bacteria may couple to one or more bacteria. Primaryadherer bacteria and/or secondary adherer bacteria may be capable ofreducing contaminants in water. In an embodiment, at least one of thesecondary adherer bacteria may reduce a greater amount of at least oneof the contaminants in water than at least one of the primary adhererbacteria.

In various embodiments, bacteria may be preserved for later use and/orfor use in systems for the reduction of contamination. Bacteria in acontainer, such as bacteria generator, may incubate and/or grow until atleast a portion of the bacteria enters the starvation phase. Thebacteria then may be preserved. In some embodiments, bacteria may bepreserved as bacteria-alginate beads and/or immobilized on hydrophobicsubstrates.

In some embodiments, when forming bacteria-alginate beads, bacteria maybe added to alginate, such as sodium alginate. The bacteria-alginatemixture or solution is added to a solution comprising metal ions andparticles may form. In other embodiments, when forming bacteriaimmobilized on hydrophobic substrates, bacteria may be incubated on ahydrophobic substrate. The hydrophobic substrate containing bacteria maybe then added to a solution that includes alginate. The alginate maypenetrate the hydrophobic substrate. Next, the hydrophobic substrate isadded to a solution comprising metal ions and the bacteria may beimmobilized on the hydrophobic substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Features and advantages of the methods and apparatus of the presentinvention will be more fully appreciated by reference to the followingdetailed description of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

FIG. 1 depicts an embodiment of a system for the reduction ofcontaminants in water.

FIG. 2 depicts an embodiment of a system that includes settlingcontainers for the reduction of contaminants in water.

FIGS. 3A-C depict embodiments of substrates for a system for thereduction of contaminants in water.

FIG. 4A depicts a representation of an embodiment of bacteria coupled tosubstrate.

FIG. 4B depicts a representation of an embodiment of bacteria coupled tosubstrate.

FIGS. 5A-B depict graphs that show the change in concentration ofbenzene over time in an embodiment of a system that includes bacteria inthe genus Gordonia.

FIG. 6 depicts a graph that shows the change in concentration ofethylbenzene over time in an embodiment of a system that includesbacteria in the genus Gordonia.

FIG. 7A depicts a graph that shows the change in concentration ofbenzene over time in an embodiment of a system that includes bacteria inthe genus Gordonia but does not include substrate.

FIG. 7B depicts a graph that shows the change in concentration ofbenzene over time in an embodiment of a system that includes bacteria inthe genus Gordonia coupled to a substrate.

FIG. 8 depicts an embodiment of a system for the reduction ofcontaminants in water.

FIG. 9 depicts an embodiment of a system for the reduction ofcontaminants in water in which fluid is recycled through multiplecontainers.

FIG. 10 depicts a graph that shows the change in concentration ofbenzene over time in an embodiment of a system in which nutrients arenot added to the system.

FIG. 11 depicts a flowchart of an embodiment of a method of preservingbacteria.

FIG. 12 depicts a graph that shows the change in concentration of methylethyl ketone over time in an embodiment of a system for the reduction ofcontaminants.

FIG. 13 depicts a graph that shows the change in concentration oftrichloroethylene over time in an embodiment of a system for thereduction of contaminants.

FIG. 14 depicts a graph that shows the change in concentration oftoluene over time in an embodiment of a system for the reduction ofcontaminants.

FIG. 15 depicts a graph that shows the change in concentration ofm-xylene over time in an embodiment of a system for the reduction ofcontaminants.

FIG. 16 depicts a graph that shows the change in concentration ofp-xylene over time in an embodiment of a system for the reduction ofcontaminants.

FIG. 17 depicts a graph that shows the change in concentration ofo-xylene over time in an embodiment of a system for the reduction ofcontaminants.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Thedrawings may not be to scale. It should be understood that the drawingsand detailed description thereto are not intended to limit the inventionto the particular form disclosed, but to the contrary, the intention isto cover all modifications, equivalents and alternatives falling withinthe spirit and scope of the present invention as defined by the appendedclaims.

DETAILED DESCRIPTION OF EMBODIMENTS

Herein are described systems and methods for the reduction ofcontaminants in water. The system may process industrial wastewater,municipal wastewater, and/or water in septic or sewer systems. Thesystem may process wastewater using bacteria to reduce the amount ofcontaminants in the wastewater. The bacteria may form a biofilm on asubstrate.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In the context of this application, the following terms aredefined as:

An “air source” refers to a device capable of providing air or othergasses to a liquid.

“Bacteria” refers to any member of the Bacteria Domain.

A “bacteria generator” refers to a device capable of allowing one ormore bacteria to grow and/or reproduce.

A “biofilm” refers to a collection of more than one bacteria coupledtogether.

A “contaminant” refers to any unwanted substance or compound.

“Coupling” refers to attaching, bonding, adhering, welding, or a directconnection of two or more objects.

“Enteric bacteria” refers to bacteria that are found in the digestivetract of animals.

A “filament” refers to a portion of a bacterium that extends from thebody of the bacterium.

“Foam” refers to an aggregate of gas bubbles formed in a liquid orsolid. Foam in a liquid may suspend solid particles and inhibit settlingof the solid particles to a bottom of a container.

A “footprint” refers to an area on a surface an object occupies.

“Gene-up regulation” refers to activation of a property of a bacteriumafter the bacterium couples to a substrate. For example, a gene may beactivated, protein synthesis may occur, and/or metabolic activity may beincreased or decreased during gene-up regulation of a bacterium.

A “heterotroph” is an organism that requires organic compounds as acarbon source for growth and development. A heterotroph is not able touse carbon dioxide as its sole carbon source.

A “hydrophobic substrate” refers to a substrate that does not formhydrogen bonds with itself, which causes it to at least partially repelwater.

An “oligotroph” refers to an organism that can live in environments witha carbon concentration of less than 1 ppm.

An “organic compound” refers to a compound that includes carbon. Anorganic compound may include elements other than carbon, such as oxygen,nitrogen, sulfur, and/or metals.

“Primary adherer bacteria” refers to any member of the Bacteria Domaincapable of coupling to a substrate and/or other bacteria.

“Stagnant regions” refers to areas that are not substantially flowing.

“Secondary adherer bacteria” refers to any member of the Bacteria Domaincapable of coupling to another bacteria but incapable of coupling to asubstrate.

“Reducing contaminants in water” refers to reducing an amount ofcontaminant in water, degrading contaminants, altering contaminants(e.g. altering a metal contaminant such that it precipitates), absorbingcontaminants, immobilizing contaminants, and/or removing one or morecontaminants from water.

“Wastewater” refers to a fluid comprising one or more contaminants.

In some embodiments, a system for the reduction of contaminants in watermay include one or more, one or more containers 100, bacteria generators200, and/or one or more air sources 300, as depicted in FIG. 1. One ormore bacteria generators 200 and/or air sources 300 may be coupled to acontainer 100. One or more air sources 300 may be coupled to bacteriagenerator 200. One or more substrates 400 may be positioned in acontainer 100. Bacteria may be coupled to the substrate 400 and/or otherbacteria in a container 100 to form a biofilm in the container. In anembodiment, bacteria may be provided to a container 100 by one or morebacteria generators 200 and/or an air bubble stream may be provided byone or more air sources 300.

In some embodiments, the system may include one or more containers. Acontainer may be formed of plastic, metal, and/or other materials. Acontainer may include one or more coatings. A coating may inhibitcorrosion and/or facilitate removal of solids from a container. Forexample, a container may have a polytetrafluoride coating to inhibitcorrosion and to inhibit solids from adhering to the container.

In some embodiments, a footprint of a container may be substantiallysquare, substantially circular, substantially oval, substantiallyrectangular, and/or irregularly shaped. A container may have a shapeconfigured to minimize stagnant regions in the container. In certainembodiments, the shape of the inner surface of the container mayminimize stagnant regions in the container during mixing. The innersurfaces of a container may be rounded instead of meeting at an edge.For example, an inner surface of a container may have a shapesubstantially similar to an oval or a circle to minimize the presence ofstagnant regions in the container, during use. In an embodiment, acontainer may have a shape in which substantially all of the liquid inone or more of the containers circulates when mixed with a stirrerduring use.

In an embodiment, a bottom surface of a container may have a shape tocollect bacteria or other solid materials in the container that sink toa bottom of the container and/or minimize the affect of solids sinkingto a bottom surface on flow within the container. For example, a bottomsurface of a container may be tapered, convex, or substantiallyconically shaped. A bottom region of a container may have approximately10% to approximately 35% grade from horizontal. In an embodiment, abottom region of a container may have approximately 23% to approximately27% grade from horizontal.

In some embodiments, a conical bottom or tapered bottom region of acontainer may facilitate the removal of solids at the bottom of thecontainer. As solids from the wastewater and/or biofilm sink to a bottomof a container, the solids may collect and inhibit contact ofcontaminants in wastewater with at least a portion of the biofilm. If abottom surface of a container is tapered, convex, or substantiallyconical, then solids may collect in the bottom region of the containerwithout substantially affecting contact between contaminants in thewastewater in the container and the bacteria of the biofilm.

In certain embodiments, a container with an elliptical inner surfacecross-section area may be used in the system. Using a container with anelliptical inner surface cross-sectional area may allow a height of thecontainer to be reduced while maintaining a similar capacity to acircular inner surface cross-sectional area container. In an embodiment,a container may have a length that is at least approximately 34% greaterthan its width and/or at least approximately 39% greater than itsheight. In another embodiment, the length of a container may be at least22% greater than the height of the container with a bottom region thatis tapered.

In some embodiments, a container may have a shape that allows enhanceduse of the top surface of a platform. It may be desirable to utilizecontainer with a footprint that maximizes area used on a surface of aplatform. For example, if a platform has a rectangular top surface, afootprint and/or inner surface of a container may be oval or oblong tomaximize an area on the top surface of a platform that the containerutilizes and minimizing stagnant areas in the container. An area of afootprint of a container may be at least three-fourths the area of a topsurface of a platform.

In an embodiment, a bottom surface of a container may have a shape tocollect bacteria or other solid materials in the container that sink tothe bottom of the container. A bottom surface of a container may have ashape such that when solids sinking to the bottom surface, fluid flowproximate the substrate in the container is not substantially inhibited.For example, a bottom surface of a container may be tapered, convex, orsubstantially conically shaped. As solids from the wastewater and/orbiofilm sink to a bottom of a container, the solids may collect andinhibit contact of contaminants in wastewater with at least a portion ofthe biofilm. If a bottom surface of a container is tapered, convex, orsubstantially conical, then solids may collect in the bottom region ofthe container without substantially inhibiting contact betweencontaminants in the wastewater in the container and the bacteria of thebiofilm.

A container may include one or more stirrers to agitate fluids and/orgasses in the container. One or more stirrers may be positioned toreduce dead mixing zones in the container. For example, a container withan oval cross-sectional area may include two stirrers approximatelyequally spaced across a bottom surface to inhibit areas of fluidstagnation the container.

In some embodiments, a container may include one or more inlets forwastewater streams, air bubble streams, and/or bacteria. A container mayinclude one or more outlets for removal of fluids and/or solids from thecontainer.

In some embodiments, filters may be coupled to inlets and/or outlets. Afilter may be coupled to an inlet to remove and/or break apart largesolids. A filter may be coupled to an outlet to prevent solids such aswaste solids, bacteria, and/or particulate matter from flowing out ofthe container. In an embodiment, a filter may inhibit contaminants fromwater flowing out of the container. For example, filter paper or anactivated carbon filter may be coupled to an outlet to removecontaminants from a stream flowing out of the container.

In some embodiments, a filter comprising activated carbon, such as agranulated activated carbon filter, may be coupled to an outlet of acontainer. An activated carbon filter may remove organic compoundsand/or metal ions. The activated carbon filter may remove fine particlesand/or bacteria from fluid flowing out of a container.

In certain embodiments, an electrocoagulation system may be coupled toinlets and/or outlets. An electrocoagulation system may be used prior toallowing fluid to enter a container comprising a biofilm and/or afterallowing fluid to leave a container that includes a biofilm. Theelectrocoagulation system may cause compounds to precipitate and floatto a top or bottom surface of a container for removal. In an embodiment,an electrocoagulation system may charge ions in a fluid. The chargedions may bind to oppositely charged ions and form a precipitate. Thenthe precipitates may float to a top surface or sink to a bottom surfaceof a container for removal from the fluid. In an embodiment theprecipitates may be filtered out of the fluid.

One or more of the containers may be settling container. As depicted inFIG. 2, the system for reduction of contaminants may include one or moresettling containers coupled to one or more other containers. A firstsettling container 105 may include a filter 110 that retains and/orbreak up large solids. The first settling container 105 may allow solidsin wastewater entering the settling container to settle to a bottom ofthe settling container and be removed via a drain 115. Wastewater fromthe settling container 105 may be introduced into a container 120containing bacteria 125 using a valve, such as a variable time releasefloat valve. The container 120 may include one or more bacteria 125 thatreduce contamination in the wastewater. In an embodiment, a secondsettling container 130 may be coupled to the container 120 containingbacteria 125 such that processed water may flow from the container thatincludes bacteria to the second settling container, in which solids fromthe container with bacteria may settle and may be removed via a drain135. A disinfectant, such as chlorine 140 may also be added to the waterin the second settling container 130. Water with substantially lesscontamination than the wastewater entering the first settling containermay exit the second settling container via one or more outlets 145. Incertain embodiments, water exiting the second settling container may berecycled through a recycle line 150 into the container 120 containingbacteria 125 until a desired level of reduction of contamination isachieved.

In some embodiments, one or more substrates may be positioned in acontainer. A substrate may be a structure on which a biofilm grows in acontainer. A substrate may be fixed to the container and/or removablefrom the container.

A substrate may be formed of plastic such as polypropylene or Kevlar®,metal, natural fibers such as cotton, other materials, or combinationsthereof. A substrate may be formed of and/or include a coating formed ofa hydrophobic material, such as polyethylene. In an embodiment, asubstrate may be a mesh commercially available from ACS Industries. Inc.(Woonsocket, R.I.). For example, a Polymer Mist Eliminator DWG No.04-15841-01, style 8PP, 74 inch outer diameter, 12 inch thick polymeshmay be used. In certain embodiments, the material selected to form thesubstrate may not substantially degrade in the presence of thewastewater to be treated.

A substrate may be planar, substantially cylindrical, substantiallyconical, substantially spherical, substantially rectangular,substantially square, substantially oval shaped, and/or irregularlyshaped. FIG. 3A depicts embodiments of different substrates. A substratemay have a shape similar to a cage, see FIG. 3B. A substrate may beknitted or woven mesh. A substrate may be corrugated and/or mesh. Forexample, a substrate may have a shape similar to an air filter. One ormore substrates may be configured to couple to each other, see FIG. 3C.

In some embodiments, one or more bacteria may couple to a substrate in acontainer to form a biofilm. In an embodiment, bacteria forming thebiofilm may not substantially slough off of the substrate, during use.The bacteria may be aerobic. Some of the bacteria may be oligotrophic,heterotrophic, enteric, and/or combinations thereof.

The bacteria may be capable of reducing contaminants in wastewater. Insome embodiments, a biofilm may be capable of significantly reducingcontaminants in water quickly. For example, wastewater may only have toreside in a container with the biofilm for less than 24 hours tosignificantly reduce an amount of contaminants in the wastewater.

One or more of the bacteria may reduce an amount of and/or degradepesticides, industrial wastewater, wastewater from septic systems,and/or municipal wastewater. In some embodiments, one or more of thebacteria may reduce an amount of and/or degrade metal compounds and/ororganic compounds such as alkanes, alkenes, aromatic organic compounds,and/or polychlorinated benzenes. Some bacteria may cleave long chainbiopolymers into monomers, which other bacteria degrade. In anembodiment, bacteria may degrade at least a portion of organic compoundsinto at least carbon dioxide and water.

In some embodiments, a biofilm may include one or more primary adhererbacteria 500 and or one or more secondary adherer bacteria 600, seeFIGS. 4A-4B. Primary adherer bacteria 500 may, be capable of coupling toone or more substrates 400 in a container and or other bacteria. Incertain embodiments, primary adherer bacteria 500 may couple with asubstrate 400 such that the primary adherer bacteria are inhibited frombeing dislodged from the substrate during use. In an embodiment, primaryadherer bacteria 500 may irreversibly couple to a substrate 400.

Primary adherer bacteria may have longitudinal and latitudinal sides. Insome bacteria, a longitudinal side may be longer than a latitudinal sideor vice versa. Primary adherer bacteria may couple to bacteria and or asubstrate along a longitudinal and or a latitudinal side. In anembodiment, a type of primary adherer bacteria may only couple to asubstrate on one of its latitudinal sides. Another type of primaryadherer bacteria may only couple to a substrate on one of itslongitudinal sides. A shape and or a density of a biofilm may becontrolled by selecting one or more types of primary adherer bacteriathat have a preference for coupling with substrate along a specificside.

In some embodiments, primary adherer bacteria 500 may include a stalk700. For example, bacteria in the genus Caulobacter have a stalk. Astalk may be a narrower than the body of the primary adherer bacteria. Astalk may be capable of coupling to inanimate objects. An end of a stalkof a primary adherer bacteria maybe couple to an inanimate object, suchas a substrate, but not couple to bacteria. For example, an end of astalk of a primary adherer bacteria may include a holdfast, such as asugar based holdfast, which allows the end of the stalk to bind with asubstrate.

In an embodiment, a stalk may grow. A stalk of a primary adhererbacteria may be capable of growing from about 5 nm to about 200 nm. Itmay be advantageous to utilize a bacteria capable of extending abiofilm. If a food source is not plentiful proximate primary adhererbacteria with stalks, the stalks may grow to position the primaryadherer bacteria in another region of the fluid with a greater foodsource.

Primary adherer bacteria 500 may include one or more filaments 750, suchas organelle, capable of coupling with other bacteria. For example,bacteria in the genus Gordonia have several filaments. Some primaryadherer bacteria may have filaments capable of coupling only with othertypes of bacteria (e.g. the filaments will not couple with the sameprimary adherer bacteria from the same genus).

In some embodiments, primary adherer bacteria may include bacteria fromthe class Actinobacteria Alphaproteobacteria, or combinations thereof.Primary adherer bacteria may include bacteria from the genus Gordonia,Caulobacter, or combinations thereof.

Secondary adherer bacteria 600 may be capable of coupling with one ormore other bacteria including primary adherer bacteria 500. In someembodiments, secondary adherer bacteria 600 may not be capable ofcoupling to a substrate 400. In an embodiment, secondary adhererbacteria may include bacteria from the class Bacilli,Gammaproteobacteria, Betaproteobacteria, or combinations thereof.Secondary adherer bacteria may include bacteria from the genus Bacillus,Pseudomonas, Zoogloea, Enterobacter, or combinations thereof.

Primary adherer bacteria and/or secondary adherer bacteria may becapable of reducing contaminants in water. Secondary adherer bacteriamay be capable of reducing a greater amount of one or more types ofcontaminants than one or more of the primary adherer bacteria. In someembodiments, sessile bacteria may experience gene-up regulation thatincreases the metabolic activity of the sessile bacteria. Sessilebacteria may have a metabolic activity 4 times the metabolic activity ofplank-tonic bacteria. Primary adherer bacteria may experience gene-upregulation of metabolic activity due to their attachment to a substrateand/or secondary adherer bacteria may experience gene-up regulation dueto their attachment to other bacteria. In an embodiment, sessile primaryadherer bacteria may experience greater gene-up regulation of metabolicactivity that sessile secondary adherer bacteria.

In some embodiments, bacteria provided to a container may be selected toreduce specific contaminants. Bacteria may be selected for their abilityto withstand a pre-determined amount of a contaminant, such as 100 ppmof aromatic organic compound, and/or fluctuations in pH. For example,bacteria selected may include bacteria from the genus Enterobacter,Pseudomonas, Gordonia, Bacillus, Agrobacterium, Caulobacter, and/orZoogloea. The biofilm may include bacteria in the genus Nocardia,Thiothrix or Beggiatoa. In an embodiment, a biofilm may includeEnterobacter cloacae, Pseudomonas putida, Pseudomonas stutzeri, Gordoniasp., Bacillus subtilis, Agrobacterium sp., Caulobacter vibrioides,and/or bacteria in the genus Zoogloea. In another embodiment, a biofilmmay be formed from a combination of bacteria, such as FreeFlow®,commercially available from NCH Corp (Irving, Tex.).

In some embodiments, the biofilm may include bacteria of the phylumActinobacteria phy. nov., class Actinobacteria, subclassActinobacteridae, order Actinomycetales, suborder Corynebacterineae,family Gordoniaceae, and/or genus Gordonia. In some embodiments,bacteria in the genus Gordonia may have filaments. The filaments may becapable of binding with a substrate and/or other bacteria. The filamentsmay promote formation of a more even biofilm. Bacteria in the genusGordonia may be capable of degrading one or more organic compounds, suchas benzene, toluene, ethylbenzene, o-xylene, p-xylene, and/or m-xylene.A system including bacteria in the genus Gordonia coupled to a substratemay be capable of reducing an amount of benzene in wastewater, see.FIGS. 5A-B which depicts a graphical representation of the removal ofbenzene by the system. A system including bacteria in the genus Gordoniathat is coupled to a substrate may reduce an amount of ethylbenzene inan aqueous solution, see FIG. 6 which depicts a graphical representationof the removal of ethylbenzene by a system.

In some embodiments, a biofilm including bacteria in the genus Gordoniamay be capable of degrading rubber compounds, desulphurize aromatics,and/or degrade pyridine compounds. Bacteria in the genus Gordonia may becapable of removing sulfur from petrochemical products. In anembodiment, bacteria in the genus Gordonia may produce biosurfactantsthat facilitate remediation and/or degradation of organic andmetal-based contamination. Biosurfactants may assist in thesolubilization of various pollutants and/or allow bacteria to morerapidly uptake pollutants for degradation or immobilization.

Bacteria in the genus Gordonia may go into a state of latency duringperiods of stress, introduction of a toxin, nutrient deprivation, and/oroxygen depravation. Bacteria in the genus Gordonia may be capable ofreviving out of the state of latency once the environment becomesconducive to the bacteria. It may be advantageous to utilize bacteriacapable of going into a latent state and reviving, so that bacteria in abiofilm may not die if the environment, such as in a bioreactor, changessignificantly.

Bacteria in the genus Gordonia may cause foaming in wastewater treatmentsystems. However, when bacteria in the genus Gordonia are coupled to asubstrate, foaming is inhibited and gene-up regulation occurs causingthe bacteria to be capable of reducing contaminants from water. FIG. 7Adepicts a graph of benzene concentrations over time in a control system(e.g., without bacteria) and in an embodiment of a system that includesbacteria in the genus Gordonia but no substrate. FIG. 7B depicts a graphof benzene concentrations over time in embodiments of a system thatincludes substrate and bacteria in the genus Gordonia and a system thatincludes a substrate and without bacteria. As shown in the graphs,bacteria in the genus Gordonia is capable of degrading more benzene whencoupled to a substrate that when the bacteria is not coupled tosubstrate. The phenomena of bacteria possessing a greater ability todegrade and/or reduce contaminants more efficiently when bound (e.g.,gene-up regulation) is not limited to bacteria in the genus Gordonia butis present in several types of bacteria. Using bacteria with increasedcontamination reduction abilities when bound allows formation of a morestabile biofilm (e.g., since bacteria are coupled to the substrate)and/or a more efficient biofilm.

In some embodiments, the biofilm may include bacteria of the phylumProteobacteria phy. nov. class Alphaproteobacteria, orderCaulobacterales, family Caulobacteraceae, and/or genus Caulobacter.Bacteria in the genus Caulobacter may convert heavy metals such asmercury, copper, cadmium, and cobalt in aqueous solutions into chemicalforms that are less toxic, less soluble, and/or precipitate out ofsolution. Some bacteria in the genus Caulobacter have resistance to someantibiotics such as chloramphenicol, tetracycline, erythromycin, andtobomycin. Resistant bacteria may be from plasmid transfer betweenantibiotic resistant intestinal or human associated bacteria found inwastewater and bacteria in the genus Caulobacter.

Bacteria in the genus Caulobacter are oligotrophs and may be capable ofsurviving in low carbon concentration environments. In some embodiments,bacteria in the genus Caulobacter may be capable of forming a uniformbiofilm due to the bacteria shape. Bacteria in the genus Caulobacterhave a motile stage characterized by a swarmer cell and a sessile stagecharacterized by a stalk shaped cell. The stalks of the bacteria in thegenus Caulobacter may grow. It may be desirable to use a bacteria with agrowing stalk since the bacteria may be better able to survive changesin environment. For example, if nutrients proximate a bacterium'slocation are depleting, then the stalk of the bacterium in the genusCaulobacter may grow and the bacterium can be positioned in a newlocation with a more nutrients.

While some bacteria are capable of forming a biofilm through thesecretion of polysaccharides, bacteria in the genus Caulobacter may becapable of forming a biofilm using a stalk. In an embodiment, usingbacteria with stalks may allow the creation of a more uniform biofilmwhen compared with a biofilm formed without the use of bacteria withfilaments. For example, a biofilm may be formed of a first layerincluding bacteria in the genus Caulobacter and one or more other layerscoupled to the bacteria in the genus Caulobacter. The stalks may becapable of coupling to the substrate but may not be capable of couplingto other bacteria. In an embodiment, bacteria in the genus Caulobactermay only couple with a substrate at the holdfast at an end of its stalk.

In an embodiment, bacteria in the genus Caulobacter are capable offrequently entering and exiting a stationary phase. It may be desirableto utilize bacteria capable of entering and exiting the stationaryphase, because the bacteria may be more durable and/or capable ofsurviving environments with fluctuations in levels of nutrients.

In some embodiments, the biofilm may include bacteria of the phylumProteobacteria phy. nov., class Gammaproteobacteria, orderEnterobacteriales, family Enterobacteriaceae, and/or genus Enterobacter.Bacteria in the genus Enterobacter may be enteric, anaerobic, and aheterotroph. Bacteria in the genus Enterobacter may produce hydrogenwhen metabolizing organic compounds. Bacteria in the genus Enterobactermay be capable of degrading aromatics, such as 2.4.6-trinitrotoluenethat is commonly found in wastewater produced in munitions production.Bacteria in the genus Enterobacter may be capable of degrading nitrateesters, such as pentaerythritol tetranitrate and glycerol trinitrate.

In some embodiments, the biofilm may include bacteria Of the phylumFirmicutes phy. nov., class Bacilli, order Bacillales, familyBacillaceae, and/or genus Bacillus. Bacteria in the genus Bacillus maybe good oligotrophs and capable of surviving in an environment with alow concentration of organic compounds. Bacteria in the genus Bacillusmay be capable of degrading organic compounds, such as organic compoundsproduced from plant and animal sources (e.g., cellulose, starch, pectin,proteins, hydrocarbons). In an embodiment, a biofilm comprising bacteriain the genus Bacillus may cleave long chain biopolymers into monomersthat are degradable by other bacteria. Bacteria in the genus Bacillusmay be cable of nitrification, denitrification, and/or nitrogenfixation. Bacteria in the genus Bacillus may be capable of fermentingcarbohydrates, producing glycerol and butanediol, producing enzymes forutilization in detergents, paralyzing insects, degrading biopolymers,and/or synthesis for use in industrial processes such as the productionof antibiotics.

In some embodiments, it may be desirable to utilize bacteria in thegenus Bacillus to create a biofilm capable of surviving in harshenvironments. Bacteria in the genus Bacillus may produce spores that arehighly resistant to stressful environments and/or toxic environments.Bacteria in the genus Bacillus may synthesize antibiotics that killproximate bacteria and cause the dead bacteria to lyse and release theircontents. Bacteria in the genus Bacillus may absorb the nutrientsreleased by the ruptured cells. This process may require less energythan forming spores.

In some embodiments, the biofilm may include bacteria of the phylumProteobacteria phy. nov., class Gammaproteobacteria, orderPseudomonadales, family Pseudomonadaceae, and/or genus Pseudomonas.Bacteria in the genus Pseudomonas may be good heterotrophs. Bacteria inthe genus Pseudomonas may be capable of degrading organic compounds,such as trichloroethylene. In an embodiment, bacteria in the genusPseudomonas may degrade monomer organic compounds. Bacteria in the genusPseudomonas may be capable of degrading aromatic organic compounds suchas toluene, xylene, naphthalene, or polynuclear aromatic organiccompounds. In certain embodiments, bacteria in the genus Pseudomonas mayprefer to degrade simple organic compounds when compared to otherorganisms.

In some embodiments, it may be desirable to include bacteria in thegenus Pseudomonas in a biofilm since they are capable of withstandingfluctuations in environment. Bacteria in the genus Pseudomonas mayproduce o-acetylated alginate that encapsulates the bacteria to protectthe bacteria from stressful environments. Bacteria in the genusPseudomonas may have filaments. The filaments may help bacteria in thegenus Pseudomonas to attach to substrates and/or other organisms. Thefilaments and production of alginate by bacteria in the genusPseudomonas may promote formation of a biofilm and/or formation of abiofilm coupled to a substrate.

In certain embodiments, the biofilm may include bacteria of the phylumProteobacteria phy. nov., class Betaproteobacteria, order Rhodocyclales,family Rhodocyclaceae, and/or genus Zoogloea. Bacteria in the genusZoogloea may be a good heterotroph. Bacteria in the genus Zoogloea maybe capable of degrading high concentrations of proteins. Bacteria in thegenus Zoogloea may produce exopolysaccharide that contributes to theability of a biofilm containing bacteria in the genus Zoogloea totolerate fluctuating, stressful, and/or toxic environments.

In various embodiments, the biofilm may include bacteria of the phylumActinobacteria phy. nov., class Actinobacteria, order Actinomycetales,suborder Corynebacterineae, family Nocardiaceae, and/or genus Norcardia;bacteria of the phylum Proteobacteria phy. nov., classGammaproteobacteria, order Thiotrichales, family Thiotrichaceae, and/orgenus Thiothrix; and/or bacteria of the phylum Proteobacteria phy. nov.class Gammaproteobacteria, order Thiotrichales, family Thiotrichaceae,and/or genus Beggiatoa. Bacteria of the suborder Corynebacterineae andbacteria of the family Thiotrichaceae may have similar behavior. Forexample, both may experience gene-up regulation of metabolic activitywhen attached to a substrate. In an embodiment bacteria of the suborderCorynebacterineae and bacteria of the family Thiotrichaceae may causefoaming in a container when planktonic.

Bacteria in the genus Gordonia may be capable of degrading one or moreorganic compounds, such as benzene, toluene, ethylbenzene, o-xylene,p-xylene, and/or m-xylene. A system including bacteria in the genusGordonia coupled to a substrate may be capable of reducing an amount ofbenzene in wastewater, see. FIGS. 5A-B which depicts a graphicalrepresentation of the removal of benzene by the system. A systemincluding bacteria in the genus Gordonia that is coupled to a substratemay reduce an amount of ethylbenzene in an aqueous solution, see FIG. 6which depicts a graphical representation of the removal of ethylbenzeneby a system.

In some embodiments, one or more bacteria generators may provide one ormore of the bacteria that form, supplement, and/or replenish the biofilmin a container. Bacteria generator may be a container capable ofincubating one or more types of bacteria. In an embodiment, bacteriagenerator may produce more than one type of bacteria simultaneously. Inanother embodiment, a system may include bacteria generator for eachstrain or set of strains of bacteria that form the biofilm. Bacteriagenerator may be a BioAmp®, commercially available from NCH Corp(Irving, Tex.). Bacteria generator may include one or more nutrientsources and/or be coupled to one or more containers such that bacteriafrom the bacteria generator is provided to the container. Bacteriagenerator may be capable of producing a predetermined amount of bacteriain less than 48 hours. In an embodiment, bacteria generator may becapable of producing a predetermined amount of bacteria in less than 24hours.

Bacteria generator may be capable of producing a constant supply ofbacteria to a container. In certain embodiments, bacteria generator mayincubate bacteria as described in U.S. Pat. No. 5,599,451 to Guiot,which is incorporated by reference. In an embodiment, bacteria generatormay facilitate rapid formation of a biofilm in a container, sincebacteria can be supplied to the biofilm to supplement growth of thebacteria in the container. Bacteria generator may be capable ofproducing different combinations and/or ratios of bacteria during use.In addition, unlike many automated bacteria incubators, the bacteriagenerator may be capable of inoculating the bacteria in the bacteriagenerator, as desired.

In some embodiments, one or more air sources may be coupled to one ormore containers and/or one or more bacteria generators to provide air oroxygen. An air source may be positioned on a top surface of a container.An air source may provide Vacuum Bubble Technology (VBT) aeration, suchan air source commercially available from Advanced Aeration. Inc(Angleton, Tex.). An air source may substantially maintain an aerobicenvironment in the container and/or the bacteria generator.

In certain embodiments, an air source may produce an air bubble stream.An air source may be configured to produce bubbles with a fixed orvariable air bubble diameter. An air source may produce air bubbles withan average bubble diameter less than approximately 1 mm. In anembodiment, an air source may produce air bubbles with an average bubblediameter less than approximately 5 mm. An air source may produce airbubbles with an average bubble diameter from approximately 0.5 mm toapproximately 0.2 mm. In another embodiment, an air source may produceair bubbles with an average bubble diameter from approximately 0.3 mm toapproximately 0.2 mm.

An air source may produce bubbles with a buoyancy less than the surfacetension of water and/or wastewater in a container. An air source mayproduce bubbles capable of remaining diffused in fluids in a containerfor approximately 6 to 12 hours rather than floating to a top surface ofthe fluid. In an embodiment, an air source may produce air bubbles in atleast a partial vacuum. In certain embodiments, an air source mayproduce bubbles filled with gas or combinations of gasses, such asoxygen, nitrogen or carbon dioxide.

In some embodiments, the air source may assist in fluid mixing in acontainer. An air bubble stream may flow from the air source with aforce sufficient to at least partially mix fluids in a container. In anembodiment, an air source may produce turbulent areas that agitatefluids in the container and/or increase contact between the bacteria andthe contaminants to allow faster reduction in the amount ofcontaminants. An air source may be coupled proximate a stirrer in acontainer to amplify the mixing provided by a stirrer in a container.

An air source that provides an air bubble stream may be preferable to aforced air diffuser, air compressor, and surface aerators that pump airinto a water column. Pumping air into a water column may form largebubbles which quickly float out of the water column. Water can onlycontain 8 molecules of oxygen per million molecules of water, thereforewhen large bubbles of air are produced and quickly travel through awater column, the probability that oxygen will diffuses into the wateris low. Thus, using small bubbles, and as a result increasing the totalsurface area of air bubbles exposed to water, increases the probabilitythat oxygen will diffuse into the water. In addition, using bubbles thatremain in fluid in the container for at least 6 hours increases theexposure time of the bubbles to the fluid and thus increases theprobability of oxygen diffusion into the fluid.

In some embodiments, a system for reduction of contaminants may includea controller. A controller may be configured to automate the system. Thecontroller may measure various parameters of the system, such aspressure; temperature; pH; amount of contaminants in a container, awastewater stream, and/or an outlet stream; an amount, type, and/orratio of types of bacteria in a container; an amount of bacteria thatsettles at a bottom of a container, an amount of solids collected at abottom of a container; flow rates of wastewater, streams of bacteriafrom bacteria generator, air bubble stream, and/or drains; and/or avolume of w ater in a container and/or bacteria generator. A controllermay use measurements of the various parameters to modify, values of oneor more parameters of the system, such as flow rates of bacteria,wastewater stream, and/or outlet stream, temperatures; pH; and/oraverage diameters of air bubbles produced in an air source. A controllermay measure and/or modify parameters of the system continuously orperiodically.

In an embodiment, a controller may determine an amount of bacteriaand/or types of bacteria in a container and alter a flow rate ofbacteria from bacteria generator into the container and/or types ofbacteria produced in bacteria generator. In another embodiment, acontroller may detect a biological oxygen demand (BOD) level and modify,a flow rate of wastewater, nutrients, and/or air bubble stream into thecontainer. A controller may detect a level of contaminants in thecontainer and allow at least a portion of the fluid in the container toflow from the container.

In some embodiments, the system may include one or more platforms. Aplatform may be movable. A platform may facilitate the transportation ofthe system. A platform may have wheels and/or be movable with aforklift.

A platform may be formed of metal, wood, fiberglass, plastic, and/or acombination thereof. A platform may have a top surface that issubstantially circular, substantially elliptical, substantially oval,substantially square, substantially rectangular, or irregularly shaped.In an embodiment, the top surface and the bottom surface of a platformmay have a similar shape and/or size.

In certain embodiments, the platform may be selected to meet shippingand/or transportation requirements.

A platform may be a 463 L pallet to comply with shipping requirements ona plane, such as a C-130.

It may be advantageous to utilize a system comprising platforms tocreate a mobile remediation system. In some embodiments, the system mayinclude one or more containers, bacteria generators, and/or air sourcespositioned in or on one or more platforms. The system may be transportedon more than one platform and be positioned at a desired location. Oncethe system is at the desired location, the components of the system(e.g., containers, bacteria generators, air sources, and/or controllers)may be coupled. In certain embodiments, more than one component may beon a platform. For example, an air source may be positioned on top of acontainer positioned in or on a platform.

FIGS. 8-9 depict embodiments of systems for the reduction ofcontaminants. One or more types of bacteria 850 may be added to bacteriagenerator 200. Nutrients 800 may be added to the bacteria generator 200.An air stream may be provided to the bacteria generator 200 from one ormore air sources 300. Bacteria in the bacteria generator 200 mayincubate and/or reproduce to generate a desired quantity and/or ratio ofbacteria. In an embodiment, a predetermined quantity of bacteriasufficient to promote production of a biofilm in a container may beproduced in less than 48 hours.

Once a predetermined amount of bacteria is produced in the bacteriagenerator 200, bacteria from the bacteria generator may be introducedinto the container 100. One or more nutrients 800 may be delivered tothe container 100 to promote the formation of a biofilm in thecontainer. An air bubble stream may be provided to the container 100from one or more air sources 300 coupled to the container. A biofilm maybe allowed to form on the substrate 400 in the container 100. In someembodiments, a biofilm may be formed in a container prior tointroduction of wastewater. In another embodiment, a biofilm may beformed in the presence of wastewater in a container.

After at least a portion of the biofilm is formed in the container 100,wastewater 900 may be introduced into the container. The biofilm mayreduce the amount of contaminants in the wastewater. The bacteria of thebiofilm may absorb, immobilize, entrap, degrade, consume, and/or modifythe contaminants in the wastewater. Nutrients 800 may be added to thecontainer 100, if the wastewater does not contain sufficient nutrientsfor the biofilm, such as when degrading volatile organic compounds. Forexample, a system that includes bacteria in the genus Gordonia coupledto a substrate may need a minimal level of nutrients (e.g., glucose orother contaminants in the wastewater) to degrade benzene, as depicted inFIG. 10 which shows a low reduction in benzene in solutions that lackother nutrients.

When the level of contaminants in the wastewater has been reduced to orbelow an allowable level of contamination, fluid 1000 may flow from thecontainer 100, see FIGS. 8-9. The fluid 1000 may flow from the container100 after residing in the container for less than approximately 20 hoursor less than approximately 8 hours. In an embodiment, one or moreadditional containers 1150 with a biofilm 1140 may process fluid 1000flowing from the container 100, as depicted in FIG. 9. Fluid flowingfrom the additional container 1150 may be recycled 1160 through acontainer 100 and/or flow from an outlet 1170 in the additionalcontainer with less contaminants than the wastewater 900. In anembodiment, industry standards, treaties, the United StatesEnvironmental Protection Agency, and/or other regulatory agency maydetermine an allowable level of contamination in the fluid. In anembodiment, wastewater may be continuously delivered to the container100 and fluids 1000 may continuously flow out of the container with lesscontaminants than the wastewater.

In some embodiments, it may be necessary to replenish and/or replace atleast a portion of the bacteria that form the biofilm. The controllermay detect a level of bacteria in the container and allow an amount ofbacteria to flow into the container from one or more bacteriagenerators. In response to measured bacteria levels in the container,the controller may modify an amount of bacteria that flows from thebacteria generator to the container and/or amounts of bacteria,nutrients, and/or air delivered to the bacteria generator and/or thecontainer.

In an embodiment, a biofilm may be replaced in response to substantiallydecreased level of bacteria in the container and/or a substantiallydecreased level of reduction of contaminants detected by the controller.In addition, since some bacteria couple strongly with substrates evenwhen dead, a substrate for a biofilm may be replaced and/or cleanedprior to forming a new biofilm in the container. A biofilm may bereplaced by removing at least a portion of a substrate and thenreplacing the removed substrate with a new or a cleaned substrate.

In an embodiment, solids in the wastewater 900 may sink to the bottom ofthe container 100. Bacteria may separate from the biofilm and/or die anduncouple from the biofilm and sink to the bottom of the container 100. Adrain 1100 in the container 100 may allow solids to be removed from thebottom of the container, during use.

During use, some systems for the reduction of contaminants and/orwastewater treatment plants may have foaming. Foaming may cause problemsin continuous operation of a wastewater treatment plant by inhibitingcontact between contaminants in the wastewater and the biofilm, alteringmixing properties of a container, and/or blocking inlets and/or outlets.Some bacteria, such as bacteria in the genus Gordonia, Nocardia,Microthrix, and/or bacteria in the family Thiotrichaceae, such asbacteria in the genus Thiothrix and Beggiatoa, may cause foaming inwastewater treatment systems. In some embodiments, one or morehydrophobic substrates may be added to a container in a wastewatertreatment plant that has foaming. The bacteria at least partiallycausing the foaming may couple to the hydrophobic substrates and foamingmay be reduced and/or inhibited. When the bacteria, such as bacteria inthe genus Gordonia, Nocardia, Microthrix, and/or bacteria in the familyThiotrichaceae, such as bacteria in the genus Thiothrix and Beggiatoa,couples to the hydrophobic substrate, gene-up regulation occurs and thebacteria is capable of reducing contaminants in water. Several othertypes of bacteria exhibit similar gene-up regulation behavior tobacteria in the genus Gordonia. In an embodiment, the bacteria, such asbacteria in the genus Gordonia, Nocardia, Microthrix, and/or bacteria inthe family Thiotrichaceae, such as bacteria in the genus Thiothrix andBeggiatoa, may couple to the substrate rather than the container becausethe hydrophobic substrate has less electrostatic interference than thecontainer. In some embodiments, the addition of a hydrophobic substrateto a wastewater treatment system with foaming may substantiallyeliminate foaming in the wastewater treatment system.

In some embodiments, a controller may monitor foaming in a wastewatertreatment plant. When foaming reaches a predetermined level, thecontroller may allow substrates to be added to the container withfoaming. In certain embodiments, the controller may automatically allowsubstrates to be added to the container with foaming that exceeds apredetermined level. In an embodiment, the controller may produce asignal indicating that foaming has reached a predetermined level and/orindicate to an operator that substrates should be added to the containerwith foaming.

It may be desirable to preserve bacteria for later use. In someembodiments, bacteria generator may be used to preserve bacteria.Preserved bacteria may be stored for as long as approximately one yearwithout substantially affecting the bacteria's capability to return to agrowth phase. In an embodiment, preserved bacteria may be stored forapproximately six months without substantially affecting the bacteria'scapability to return to a growth phase. Preserved bacteria may be usedfor a variety of purposes including in systems for the reduction ofcontaminants in wastewater. In an embodiment, preserved bacteria may besupplied to bacteria generator to produce a desired amount of bacteriato create and/or to supplement a biofilm.

FIG. 11 depicts a flowchart of an embodiment of preserving bacteria. Oneor more types of bacteria are incubated and allowed to grow and/orreproduce in the presence of one or more nutrients 1200. In anembodiment, bacteria may be incubated and reproduce in one or morebacteria generators. The flow of nutrients is then terminated 1300 andthe bacteria are allowed to enter a starvation phase 1400. In anembodiment, the starvation phase for the bacteria may be identified bydetermining when exponential growth of the bacteria has ended. Thechange in the number of bacteria may be monitored spectroscopically. Thebacteria in the starvation phase may then be preserved 1600.

In some embodiments, the bacteria may be inoculated prior topreservation. Bacteria in the starvation phase produce stress proteinsthat protect the bacteria from shock. Therefore, when bacteria areinoculated, a greater percentage of bacteria in the starvation phasewould be able to survive the shock due to the increased production ofstress proteins. Stressing bacteria prior to preservation may allowhardier bacteria to survive the stress of inoculation while the weakerbacteria may die during inoculation. Therefore, it may be advantageousto stress bacteria prior to preserving the bacteria, since the shock mayonly allow hardier bacteria to be preserved.

It may be advantageous, in some embodiments, to preserve bacteria in thestarvation phase. The starvation phase occurs during the stationaryphase of bacteria. During the starvation or stationary phase, the rateof change of the number of bacteria is approximately constant since thenumber of bacteria generated is approximately the same number ofbacteria that die. Using bacteria in the starvation phase may also bedesirable, since when starved bacteria are introduced into anenvironment with nutrients, the bacteria are hungrier and morecompetitive for the available carbonaceous material.

In some embodiments, bacteria in the starvation phase may be preservedas bacteria-alginate beads, where the bacteria is immobilized in a bead.To produce bacteria-alginate beads, bacteria is mixed with an alginate,such as sodium alginate. In an embodiment, alginate is added to anaqueous solution including the bacteria in the starvation phase. Inanother embodiment, bacteria in the starvation phase may be added to anaqueous alginate solution. The sodium alginate or a viscous aqueoussolution containing alginate may be autoclaved at a temperature fromapproximately 115° C. to approximately 125° C. The bacteria-alginatemixture is stirred. The viscosity of the bacteria-alginate mixture mayincrease while stirring. The bacteria-alginate mixture is then added toan aqueous solution containing calcium ions.

In an embodiment, the bacteria-alginate mixture is added in drops to theaqueous solution containing calcium ions. Bacteria-alginate particlesare allowed to form in the calcium ion solution. The bacteria-alginateparticles may be firm and not as compressible as a gelatinous substance.The bacteria-alginate particles may be separated from the solutionand/or dried. The bacteria-alginate particles may be filtered from thesolution in an aseptic environment. The preserved bacteria-alginateparticles may be stored until needed and/or used in bacteria generatorin a system for the reduction of contaminants in water. In anembodiment, when the bacteria-alginate particles are revived in asolution of nutrients, the bacteria may consume and/or degrade thealginate portions of the particle.

The size and shape of the bacteria-alginate particles may becontrollable. The amount of bacteria-alginate mixture added or droppedinto the calcium solution may control the size of the particles formed.The bacteria-alginate mixture may be sprayed onto the aqueous solutioncontaining calcium ions to produce small substantially spherical-shapedparticles. Particles that are substantially cubic, pyramidal, conical,or irregularly shaped may also be formed.

In other embodiments, bacteria in the starvation phase may be preservedon hydrophobic substrates. To produce immobilized bacteria in thestarvation phase on a hydrophobic substrate, bacteria may incubate in asolution containing one or more hydrophobic substrates until thebacteria are in the starvation phase. Alginate is mixed in an aqueoussolution and may be autoclaved at a temperature from approximately 115°C. to approximately 125° C. The hydrophobic substrate that includes thebacteria in the starvation phase may then be introduced into thealginate solution. Alginate may at least partially saturate thehydrophobic substrate. The hydrophobic substrate then may be contactedwith an aqueous solution containing calcium ions. The hydrophobicsubstrate may be separated from the solution and/or vacuum filtered. Thehydrophobic substrate may be allowed to dry. In certain embodiments, thehydrophobic substrate containing preserved bacteria in the starvationphase may be stored until needed, used in bacteria generator in a systemfor reduction of contaminants in water, and/or added to a container toform a biofilm.

Although adding bacteria-alginate mixture to calcium ions is described,other metal ions solutions may be used successfully as well, includingbarium, copper, or zinc metal ion solutions. It may be desirable to usea calcium ion solution because calcium is available at a low cost fromsources such as limestone and/or calcium is not generally considered acontaminant, unlike copper or zinc.

Preserving bacteria in particles or immobilizing bacteria on hydrophobicsubstrates may allow the preserved bacteria to be more resilient toenvironmental stress and or toxins and or may reduce cell mortality uponrevival. Unlike when using preservation methods currently known in theart, such during lyophilization or the formation of compressed tablets,the bacteria are not dried to desiccation when bacteria are in particlesor immobilized on substrates. Although lyophilized bacteria andcompressed pellet bacteria have long shelf lives, it may take a longperiod for the bacteria to acclimate to surroundings and return to anexponential growth stage. Bacteria in particles and immobilized onsubstrates may become physiologically active within a shorter period oftime since the cells do not have to be hydrated since they were notdesiccated to the same extent during preservation.

In some embodiments, the preserved bacteria in particles and orhydrophobic substrate may be added to bacteria generator to producebacteria for a container in a system for the reduction of contaminantsin wastewater. The preserved bacteria may be revived from the starvationphase and enter exponential growth phase when introduced into an aqueoussolution containing nutrients. The preserved bacteria may consume thealginate in the particle and or hydrophobic substrate. After a period ofincubation, the bacteria may then be introduced into a container to formand/or replenish a biofilm. In an alternative embodiment, preservedbacteria in or on hydrophobic substrate may be added directly to acontainer to form a biofilm.

It is to be understood the invention is not limited to particularsystems or biological species described which may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification, the singular forms “a”,“an” and “the” include plural referents unless the content clearlyindicates otherwise. Thus, for example, reference to “a substrate”includes a combination of two or more substrates and reference to “aprimary adherer bacteria” includes mixtures of primary adherer bacteria.

Certain U.S. patents have been incorporated by reference. The text ofsuch U.S. patents is, however, only incorporated by reference to theextent that no conflict exists between such text and the otherstatements and drawings set forth herein. In the event of such conflict,then any such conflicting text in such incorporated by reference U.S.patents is specifically not incorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

EXAMPLES Example 1 Producing Bacteria in the Starvation Phase

Bacteria was incubated in a nutrient broth at a temperature of fromapproximately 25° C. to approximately 30° C. depending on which bacteriais being preserved. Bacteria in the genus Agrobacterium, Bacillus,Caulobacter, Enterobacter, Gordonia, Zoogloea and Pseudomonas wereincubated at 30° C. Bacteria in the genus Agrobacterium and Zoogloeawere incubated at 26° C. The bacteria were allowed to incubate for 24 to72 hours without the addition of an additional amount of nutrients.Bacteria in the genus Agrobacterium, Bacillus, Enterobacter, andPseudomonas were incubated for 24 to 48 hours. Bacteria in the genusCaulobacter and Gordonia were incubated for 48 to 72 hours. Bacteriawere spectroscopically monitored to determine when exponential growthceases and bacteria have entered the starvation phase.

Example 2 Producing Bacteria-Alginate Particles

40 g of sodium alginate was mixed into an aqueous solution to formsolution more viscous than water. The alginate solution was autoclavedat 121° C. for 30 minutes. The alginate solution was then allowed tocool. 500 ml of bacteria in the starvation phase, prepared according toExample 1, was added to the alginate solution to form bacteria-alginatemixture. The bacteria-alginate solution was agitated. Thebacteria-alginate solution was added in drops into 2 L of 0.55 M calciumchloride solution. The calcium chloride solution was mixed continuously.Particles, with a length and a width of approximately 5 mm, formed inthe calcium chloride solution. The particles were then filtered under atleast a partial vacuum using Whatman 40 filter paper, commerciallyavailable from Whatman (Middlesex, United Kingdom). The particles werethen dried and stored.

Example 3 Producing Bacteria Immobilized on a Hydrophobic Substrate

40 g of sodium alginate was stirred in an aqueous solution. As thesolution was stirred, the sodium alginate gelled and became too viscousto stir with a magnetic stir bar. The alginate solution was autoclavedat 121° C. for 30 minutes and then allowed to cool for at least 12hours. Bacteria, prepared according to Example 1, were incubated with anAmerican Copper Sponge mesh, commercially available from ACS Industries(Woonsocket, R.I.). The bacteria became embedded in the hydrophobicsubstrate. The bacteria-hydrophobic substrate was placed in the cooledalginate solution and allowed to be saturated with the alginate. Thebacteria-alginate-hydrophobic substrate was then added to a solutioncontaining 0.55 M calcium chloride solution. The substrate remainedsubmerged in the calcium chloride solution for 5 minutes. The substratewas then separated from the solution using aseptic techniques. Thesubstrate containing immobilized bacteria was then dried and stored.

Example 4 Contamination Reducing Systems

Bacteria mixture was prepared according to Example 1 containingEnterobacter cloacae, Pseudomonas putida, Pseudomonas stutzeri, Gordoniasp., Bacillus subtilis, Agrobacterium sp., Caulobacter vibrioides, andbacteria in the genus Zoogloea.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and methyl ethyl ketone. FIG. 12depicts a graph of the concentration of methyl ethyl ketone over time.The bacteria coupled to a substrate reduced the amount of methyl ethylketone in the solution.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and trichloroethylene. As depicted inFIG. 13, trichloroethylene was degraded by a system comprising bacteriacoupled to a substrate.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and toluene. FIG. 14 depicts a graphof the concentration of toluene over time. The bacteria coupled to asubstrate reduced the amount of toluene in the solution.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and m-xylene. As depicted in FIG. 15,m-xylene was degraded by a system comprising bacteria coupled to asubstrate.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and p-xylene. As depicted in FIG. 16,p-xylene was degraded by a system comprising bacteria coupled to asubstrate.

A portion of the prepared bacteria mixture was added to a solutioncontaining a substrate, nutrients, and o-xylene. As depicted in FIG. 17,o-xylene was degraded by a system comprising bacteria coupled to asubstrate.

1. A biofilm for treatment of wastewater comprising: one or more primaryadherer bacteria capable of coupling to a substrate; one or moresecondary adherer bacteria capable of coupling to at least a portion ofone or more of the primary adherers; and wherein at least one of theprimary adherer bacteria and/or secondary adherer bacteria is capable ofreducing a greater amount of at least one contaminant in wastewater inthe sessile state than in the planktonic state. 2-16. (canceled)
 17. Thebiofilm of claim 1, wherein the primary adherer bacteria comprises:bacteria in the genus Gordonia; and bacteria in the genus Caulobacter.18. (canceled)
 19. The biofilm of claim 1, wherein the secondary adhererbacteria comprises: bacteria in the genus Pseudomonas; bacteria in thegenus Bacillus; bacteria in the genus Enterobacter; and bacteria in thegenus Zoogloea; and bacteria in the genus Agrobacterium.
 20. (canceled)21. The biofilm of claim 1, wherein at least one of the primary adhererbacteria comprises a stalk capable of coupling with the substrate.22-23. (canceled)
 24. The biofilm of claim 1, wherein at least one ofthe primary adherer bacteria comprises filaments capable of couplingwith other bacteria and/or the substrate. 25-26. (canceled)
 27. Thebiofilm of claim 1, wherein at least one of the primary adherer bacteriacomprises bacteria capable of binding with a hydrophobic substrate. 28.The biofilm of claim 1, wherein at least one of the primary adhererbacteria comprises bacteria capable of coupling with a substrate suchthat the bacteria is inhibited from being dislodged from the substrateduring use. 29-31. (canceled)
 32. The biofilm of claim 1, wherein atleast one of the secondary adherer bacteria comprise filaments. 33-76.(canceled)
 77. A method of treating wastewater comprising: allowing oneor more primary adherer bacteria to reproduce in one or more bacteriagenerators, wherein a primary adherer bacteria is capable of coupling toa substrate; allowing one or more secondary adherer bacteria toreproduce in one or more of the bacteria generators, wherein a secondaryadherer bacteria is capable of coupling to at least a portion of aprimary adherer bacteria, and wherein the secondary adherer bacteria iscapable of reducing contaminants in water during use; allowing a biofilmcomprising one or more of the primary adherer bacteria and one or moreof the secondary adherer bacteria to form in a container.
 78. The methodof claim 77, further comprising introducing wastewater into thecontainer. 79-111. (canceled)
 112. A method for reducing foaming in awastewater treatment system comprising: introducing one or morehydrophobic substrates in one or more containers, wherein one or more ofthe containers comprise one or more bacteria, and wherein at least oneof the bacteria comprises bacteria in the order Actinomycetales and/orbacteria in the order Thiotrichales, and wherein at least one of thebacteria is configured to reduce contaminants in water; and reducingfoaming in at least one of the containers by allowing at least a portionof the bacteria in the order Actinomycetales and/or bacteria in theorder Thiotrichales to couple to at least one of the hydrophobicsubstrates
 113. The method of claim 112, wherein at least one of thebacteria in the order Actinomycetales is in the suborderCorynebacterineae.
 114. The method of claim 112, wherein at least one ofthe bacteria in the order Thiotrichales is in the family Thiotrichaceae.115. The method of claim 112, wherein at least one of the bacteria inone or more of the containers is of the genus Gordonia, Nocardia,Thiothrix, Beggiatoa, or a combination thereof. 116-130. (canceled) 131.The method of claim 112, wherein the wastewater treatment systemcontinues to operate during introduction of one or more of thehydrophobic substrates into the system.
 132. The method of claim 112,wherein foaming in the container is substantially reduced orsubstantially eliminated by allowing at least a portion of the bacteriain the order Actinomycetales and/or bacteria in the order Thiotrichalesto couple to at least one of the hydrophobic substrates.
 133. (canceled)134. The biofilm of claim 1, wherein one or more of the secondaryadherer bacteria comprises bacteria in the genus Pseudomonas, Bacillus,Agrobacterium, Enterobacter, Zoogloea, Gordonia, Caulobacter, or acombination thereof.
 135. The biofilm of claim 1, wherein one or more ofthe secondary adherer bacteria comprises Pseudomonas stutzeri,Pseudomonas putida, Bacillus subtilis, Agrobacterium sp., Enterobactercloacae, Zoogloea sp, Gordonia sp., Caulobacter vibrioides, orCaulobacter crescentus, or a combination thereof.
 136. The method ofclaim 77, further comprising providing bacteria in the genusPseudomonas, Bacillus, Agrobacterium, Enterobacter, Zoogloea, Gordonia,Caulobacter, or a combination thereof to at least one of the bacteriagenerators.
 137. The method of claim 77, further comprising providingPseudomonas stutzeri, Pseudomonas putida, Bacillus subtilis,Agrobacterium sp., Enterobacter cloacae, Zoogloea sp, Gordonia sp.,Caulobacter vibrioides, or Caulobacter crescentus, or a combinationthereof to at least one of the bacteria generators.